基于滚环扩增技术耦合切刻内切酶信号放大技术的新型超灵敏miRNA-122荧光传感器

Abstract

MicroRNA-122 (miRNA-122)的异常表达与肝癌的发生、发展及转移紧密相关，发展简单、快速、高灵敏和高特异性的miRNA-122检测方法对于肝癌的早期诊断和预后评估具有非常重要的意义。本文报道了一种基于滚环扩增技术(RCA)耦合切刻内切酶信号放大(NESA)技术的新型荧光传感器并用于超灵敏和高选择性检测miRNA-122。该方法涉及了两个阶段的反应机制，第一阶段，目标物激活切刻内切酶辅助的聚合反应，用以实现目标物与次级目标物循环放大以及生成RCA引物；第二阶段，利用RCA耦合NESA反应，实现信号传导以及多重信号放大。实验结果证明，该传感器表现出对miRNA-122的非常高的灵敏度和特异性，检测下限达到3.9 aM，较之前报道的方法，灵敏度有明显的提高。另外，该方法整个过程只需一步反应，具有操作简便、成本低、检测时间短的优势。所以，所提出的基于RCA耦合NESA的荧光传感器有望建立一个简单而实用的生物传感新平台，并应用于miRNA检测以及癌症的早期诊断和预后评估。 Aberrant expression of microRNA-122 (miRNA-122) is associated with the occurrence, develop-ment and metastasis of hepatocellular carcinoma. It’s of great importance for the development of sensitive and selective method for the detection of miRNA-122 to the early diagnosis and prognosis of hepatocellular carcinoma. A novel fluorescent biosensor has been constructed for ultrasensitive and high specific detection of miRNA-122 based on rolling circle amplification (RCA) coupled with nicking endonuclease signal amplification (NESA). Our assay involves of two stages of reaction mechanism, the first stage is target-activated nicking endonuclease-assisted polymerization, which is used for the realization of the recycle amplification of target and secondary target and the produce of RCA primer. The second stage is to achieve signal transduction and multiple signal amplification by the utilization of RCA coupled with NESA. The results reveal the constructed biosensor display high sensitivity and specificity for miRNA-122 detection, and the limit of detection is as low as 3.9 aM, which is significantly improved compared to the previous methods. Moreover, the proposed method has the advantages of rapidness, low cost, simplicity with only one-step operation. Therefore, the developed RCA coupled with NESA-based fluorescent biosensor might create a simple and practical platform for the detection of miRNA and the early diagnosis and prognosis of cancer.